NO-releasing aspirin (NO-ASA), a promising colon cancer chemopreventive agent, consists of a traditional aspirin (ASA) molecule to which -NO2, the NO-releasing moiety, is covalently attached via a chemical spacer molecule. NO-ASA inhibits colon cancer cell growth more potently than ASA by inhibiting cell proliferation and enhancing cell killing. Very little is known about the molecular targets in the cancer cell that are responsible for this effect. Our hypothesis, based on preliminary results, is that NF-kappaB is an important mediator of the effect of NO-ASA in colon cancer chemoprevention. Therefore, the goal of this study is to elucidate the molecular mechanisms by which NO-ASA modulates the NF-kappaB signaling system and to determine whether such modulation is critical for NO-ASA's chemopreventive effect against colorectal cancer. We will pursue 3 specific aims. Specific Aim 1: Assess, in human colon cancer cell lines, the effect of NO-ASA on NF-kappaB activation and correlate this event with the effect of NO-ASA on cell kinetics. Specific Aim 2: Evaluate which portion of the NO-ASA molecule is critical for its effect on NF-kappaB activation. The individual contribution of each structural component of NO-ASA will be tested: ASA, ASA+ Spacer (NO-ASA without the -NO2 group); spacer (the part of NO-ASA linking ASA to -NO2); deacetylated NO-ASA (NO-ASA minus the acetyl group). Specific Aim 3: Test the biological relevance of our in vitro findings using a preclinical model of colon cancer. We will evaluate directly in rats treated with azoxymethane, an established model of colon cancer, the role of NF-kappaB in colon cancer prevention and will test the key mechanistic findings from the in vitro study. Demonstrating that NO-ASA induces an important molecular change in the malignant colonocyte that may be relevant to its pharmacological actions in cancer would have important implications for colon cancer prevention. The results of the proposed studies not only will be of mechanistic interest but will also reveal opportunities for the rational design of drug combinations that will enhance the potency and efficacy of NO-ASA. Thus, the overall contribution of our proposal to cancer chemoprevention and control is expected to be both direct and significant.